Created by: Rio Jati Kusuma

2015

Total RNA Extraction from Dairy Product

Total RNA Extraction from Yogurt

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Weight 0.5 gram of yogurt

Dilute it with 2 mL of sodium citrate 50%
Homogenize the sample by vortexing for 30 seconds
Add 1 L of synthetic microRNA into the mixture and mix by pipetting up and down
Centrifuge for 5 minutes at 9,000 x g at 40 C
Aspirate 500 L of clear supernatant and add 2 mL of RNA lysis buffer containing DNAse I
and 500 L buffer AE (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0)
7. Mix by vortexing rigorously for 10-15 seconds
8. Aspirate 500 L into new tube and add 350 L buffer N3 (4.2 M Gu-HCl, 0.9 K-acetate, pH
4.8)
9. Centrigufe for 12 minutes at 13,000 x g at 40 C
10. Transfer the clear supernatant into new tube for RNA precipitation
Total RNA Extraction from Cheese
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Weight 10 gram of cheese

Dilute with 5 mL of sodium citrate 50%
Homogenize using mechanical waring blender
Centrifuge for 5 minutes at 9,000 x g at 40 C
Aspirate 500 L of clear supernatant and add 2 mL of RNA lysis buffer containing DNAse I
and 500 L buffer AE (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0)
6. Add 1 L of synthetic microRNA into the mixture and mix by pipetting up and down
7. Mix by vortexing rigorously for 10-15 seconds
8. Aspirate 500 L into new tube and add 350 L buffer N3 (4.2 M Gu-HCl, 0.9 K-acetate, pH
4.8)
9. Centrigufe for 12 minutes at 13,000 x g at 40 C
10. Transfer the clear supernatant into new tube for RNA precipitation
Total RNA Extraction from Yogurt (Modified Method I)
1. Weight 0.1 gram of yogurt
2. Dilute it with 250 L of buffer P1 with DNAse I added and 20 L proteinase K
3. Homogenize the sample
4. Add 200 L buffer P2 and mix by inverting the tubes 4-5 times
5. Add 350 L buffer N3 and mix by inverting the tubes 10-12 times
6. Incubate at 560 C for 10 minutes
7. Centrifuge 10,000 x g for 12 minutes
8. Transfer the clear supernatant into a new tube
9. Proceed to RNA isolation using kit
Total RNA Extraction from Yogurt (Modified Method II)
1. Weight 0.1 gram yogurt

Created by: Rio Jati Kusuma

2015

2. Dilute it with 1 mL of Trizol and homogenize by vortexing 12 seconds

3. Incubate at 600 C for 5 minutes
4. Add 200 L of chloroform and shake the tube vigorously by hand for 15 seconds
5. Incubate for 2-3 minutes at room temperature
6. Centrifuge the sample at 12,000 x g for 15 minutes at 40 C
7. Carefully, transfer the aqueous phase (the upper layer) into a new tube
8. Add 1 volume of ethanol 70% and mix thoroughly by vortexing
9. Transfer 700 L of supernatant into RNA spin coloumn
10. Centrifuge for 30 seconds at 10,000 rpm and discard flow through
11. Repeat again if the volume of the sample is more than 700 L
12. Add 500 L buffer RPE to the spin column
13. Centrifuge for 30 seconds at 10,000 rpm and discard flow through
14. Add 500 L of ethanol 80% to the spin column
15. Centrifuge for 2 minutes at 10,000 rpm and discard flow through
16. Centrifuge again for 5 minutes at 10,000 rpm to completely remove the ethanol
17. Place the spin column into new tube
18. Add 20 L of RNA free water into the middle of spin column
19. Incubate for 1 minute
20. Centrifuge at max speed for 1 minute
21. Measure the RNA quality using nanodrop